An assay of drug-induced emesis in the squirrel monkey (Saimiri sciureus)

Abstract

Background:

Emesis has significant evolutionary value as a defense mechanism against ingested toxins; however, it is also one of the most common adverse symptoms associated with both disease and medical treatments of disease. The development of improved antiemetic pharmacotherapies has been impeded by a shortage of animal models.

Methods:

The present studies characterized the responses of the squirrel monkey to pharmacologically diverse emetic drugs. Subjects were administered nicotine (0.032–0.56 mg/kg), lithium chloride (150–250 mg/kg), arecoline (0.01–0.32 mg/kg), or apomorphine (0.032–0.32 mg/kg) and observed for emesis and prodromal hypersalivation.

Results:

Nicotine rapidly produced emesis and hypersalivation. Lithium chloride produced emesis with a longer time course without dose-dependent hypersalivation. Arecoline produced hypersalivation but not emesis. Apomorphine failed to produce emesis or hypersalivation.

Conclusions:

The squirrel monkey is sensitive to drug-induced emesis by a variety of pharmacological mechanisms and is well-positioned to examine antiemetic efficacy and clinically important side effects of candidate antiemetic pharmacotherapies.

1 |. INTRODUCTION

Emesis, the forceful oral expulsion of gastrointestinal contents, has significant evolutionary value as a defense mechanism against ingested toxins.¹ However, it is also one of the most common adverse symptoms associated with both disease and medical treatments of disease. Emesis often occurs following chemotherapy, radiation, anesthesia, and surgical recovery. In addition, it has been estimated that over one third of all medications can produce emesis² and this side effect is often a dose-limiting factor.³ Not only does emesis dramatically impact quality of life, but it also negatively affects nutrition, decreases patient compliance, and delays recovery.^4,5 Chemotherapy represents a particularly striking example as many of the most common treatment regimens produce emesis in virtually all patients within 1–2 hours without prophylactic treatment.⁶ Cancer patients consistently rank emesis as among the most distressing events following chemotherapeutic treatment.^3,7,8

The need for improved antiemetics remains an important public health priority.⁹ Several drug classes have demonstrated antiemetic efficacy in preclinical and clinical studies, such as serotonin 5-HT3 receptor antagonists,^10,11 neurokinin (NK-1) receptor antagonists,^12,13 dopamine receptor antagonists,^14–16 and cannabinoid receptor agonists.^17–19 However, available antiemetics lack broad efficacy against the heterogeneous conditions by which emesis is produced. Current clinical guidelines allow for combinations of 2–3 antiemetics from different drug classes to control emesis,^20–22 but a large number of patients, including 30%−50% of patients receiving highly emetogenic chemotherapy, continue to experience symptoms despite guideline-directed prophylactic treatment.^23,24

The development of improved antiemetic therapeutics and the elucidation of the neural mechanisms underlying the emetic reflex have been hampered by a paucity of laboratory models.^25–27 Perhaps the most significant impediment is related to the fact that several of the most common laboratory animals, including the mouse, rat, guinea pig, and rabbit, are physically incapable of vomiting due to a complex array of neural and anatomical constraints.²⁸ As a consequence, preclinical studies in rodents have relied on malaise-related behavior thought to be associated with emesis, such as conditioned taste aversion²⁹ and conditioned gaping³⁰; however, these indirect measures in animals that lack the emetic reflex may have limited predictive validity.³¹ Other small mammalian species that exhibit the emetic reflex have served as laboratory subjects in the preclinical assessment of candidate antiemetics, including the ferret^14,32,33 and shrew.^17,34,35 Studies using these species have provided valuable insights related to antiemetic drug development, such as those that advanced the serotonergic treatment of chemotherapy-induced vomiting in the 1990s.³⁶ However, the relatively sparse body of work in the ferret and shrew within the larger biomedical research community complicates the incorporation of findings in these species within a multidisciplinary program necessary to address the emesis brought on by various disease states and treatment conditions.

An alternative approach could include the study of non-human primates, which, like rodents, have a long-standing and rich history of contributions to nearly every branch of biomedical science and have informed a variety of disease states as well as medication development across drug classes.³⁷ Considering these broad extant contributions and the fact that all non-human primates have an emetic reflex that is ostensibly similar to humans, it is surprising that there have been no published empirical validations of non-human primate models of emesis expressly designed to develop improved antiemetics. The aim of the present studies was to fill this knowledge gap by examining the emetic response in the squirrel monkey (Saimiri sciureus). Well-established behavioral and pharmacological methodologies in this species have provided a rigorous approach to the study of psychoactive drugs for over 60 years, including investigations of abuse liability, subjective effects, cognitive impairment, and structural, functional, and neurochemical consequences of acute and chronic drug exposure.^38–46 In addition, this species is small compared to other common laboratory non-human primates, such as macaques, resulting in relatively simplified husbandry and handling. They also carry a relatively low risk of transmitting zoonotic infections to researchers.⁴⁷ The squirrel monkey is therefore well-positioned to evaluate the emetic and antiemetic potential of drug treatment while simultaneously incorporating findings into a comprehensive in vivo drug profile, thus providing a more effective assessment of the overall translational value and clinical safety of candidate antiemetics.

Importantly, however, one cannot assume cross-species continuity in the ability of a particular drug or toxin to produce emesis, even among emetic species.^31,48,49 Apomorphine, the non-selective dopamine receptor agonist, provides an illuminating example. For decades, apomorphine has been the gold standard emetic for dogs in veterinary settings⁵⁰ and is also a potent emetic in humans.⁵¹ However, as first noted by Brizzee et al,⁵² apomorphine, delivered intravenously or subcutaneously up to doses that produce convulsions (25 and 100 mg/kg, respectively), does not elicit emesis in either the rhesus or cynomolgus Old World monkey. Curiously, apomorphine is a potent emetic in the marmoset, a small New World primate. Ando et al⁵³ reported vomiting in the marmoset following administration of 0.5 mg/kg apomorphine, and our laboratory has also documented vomiting in the marmoset minutes after administration of 0.1 mg/kg apomorphine (unpublished observations). Taken together, this underscores a cross-species disorder in drug-induced emetic responses among primates, despite the evolutionary contiguity often assumed of primitive reflexes. Moreover, it highlights the necessity of validating a drug’s emetic liability in the particular laboratory subject of interest, and without guarantee of translation to humans.

The present studies characterized the effects of several drugs with emetic potential in the squirrel monkey to appraise their suitability to contribute to antiemetic research in this non-human primate species. Emesis was measured following administration of four pharmacologically diverse agents. Nicotine, the nicotinic acetylcholine receptor agonist, and lithium chloride (LiCl), the prototypical toxin for producing taste aversion, were studied as they represent two of the most common non-chemotherapeutic emetics employed in previous work with laboratory animals, including the shrew and ferret.^54–56 Apomorphine, the non-selective dopamine agonist and common veterinary emetic summarized above, was studied to determine where the squirrel monkey resides among other primate species in response to this drug. Finally, arecoline, a muscarinic acetylcholine agonist, was investigated because it has a mechanism of action at the receptor opposite to that of the prototypical motion sickness medication and muscarinic acetylcholine antagonist, scopolamine.⁵⁷ Hypersalivation was also measured following administration of each drug, as it is thought to be a prodromal sign associated with emesis.²⁷ To further examine their emetic profile, drugs that produced emesis in the squirrel monkey were then studied under pre-feeding conditions with the expectation that recent food consumption would potentiate emetic responses.

2 |. METHODS

2.1 |. Humane care guidelines

The protocol for the present studies was approved by the Institutional Animal Care and Use Committee at McLean Hospital in a facility licensed by the United States Department of Agriculture and in accordance with guidelines provided by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources, Commission on Life Sciences.⁵⁸

2.2 |. Subjects

Four adult male squirrel monkeys (Saimiri sciureus) weighing between 0.8 and 1.0 kg served in the present studies. All subjects were experimentally and drug-naïve at the start of the studies. Subjects were housed in a temperature- and humidity-controlled vivarium with a 12-hour light/dark cycle (lights on at 7 AM). Environmental enrichment was provided daily. Subjects had unlimited access to water in the home cage and were maintained at approximate free-feeding weights by daily feedings of nutritionally balanced high-protein biscuits (Purina Monkey Chow, St. Louis, MO) and fresh fruit. Experimental sessions were conducted five days a week (M-F). With the exception of the pre-feeding studies (described below), subjects were fed 2 hours following each experimental session.

2.3 |. Apparatus

A custom-designed dual-compartment observation chamber was used to monitor two subjects simultaneously. Two clear Plexiglas cubes (25 × 25 × 25 cm) separated by an opaque Plexiglas divider resided in a light- and sound-attenuating ventilated enclosure (75 × 60 × 50 cm). Mirrors were affixed to the walls and floor of the enclosure to provide a view of orofacial and abdominal movements when subjects were facing away from the observer. White noise was present in the experimental room to provide masking sound. Subjects were leashed but otherwise unrestrained within the observation chamber.

2.4 |. Experimental procedures

During experimental sessions conducted at approximately the same time each day, subjects were placed in the observation chamber for behavioral assessment. Drug administration sessions were conducted no more than twice weekly, and control sessions, in which 0.1–0.3 mL of 0.9% saline solution was administered, were conducted during intervening days to preclude the development of conditioned responses to injection or to the observation chambers.

2.4.1 |. Assessment of drug-induced emesis and hypersalivation in squirrel monkeys

To determine the ability of drug treatment to induce emesis and hypersalivation in squirrel monkeys, doses of nicotine (0.032–0.56 mg/kg), LiCl (150–250 mg/kg), apomorphine (0.032–0.32 mg/kg), and arecoline (0.01–0.32 mg/kg) were administered in a quasi-random order. Initial assessments were conducted with each drug using 60-minute observation periods to identify a sufficient interval to capture all observable responses for each drug studied. Following these initial assessments, it was determined that subsequent observation periods of 20 minutes for nicotine, 30 minutes for arecoline, and 60 minutes for LiCl and apomorphine were sufficient.

2.4.2 |. Effects of pre-feeding on nicotine- and LiCl-induced emesis and hypersalivation

To assess whether pre-feeding could modify nicotine- and LiCl-induced emesis and hypersalivation, subjects were fed their daily meal portion 1 hour prior to drug administration (instead of approximately 21 hours prior, as outlined above). The effects of the emetic doses of nicotine (0.1, 0.32, 0.56 mg/kg) and LiCl (200, 250 mg/kg) were re-determined under these conditions.

2.5 |. Drugs

(−)-Nicotine hydrogen tartrate, apomorphine, and arecoline were purchased from Sigma-Aldrich (St. Louis, MO). LiCl was purchased from Fisher Scientific (Hampton, NH). Nicotine, apomorphine, and arecoline were prepared in 0.9% saline solution. LiCl was prepared in sterile water. The pH of nicotine was adjusted to ~7.0 with the addition of 0.1 N sodium hydroxide as needed. All drugs were administered via intramuscular (im) injection in volumes of 0.4 mL/kg or less. Drug concentrations are expressed in terms of their free base.

2.6 |. Data analysis

During experimental sessions, subjects were monitored continuously in the observation chamber. Instances of licking lasting longer than 2 seconds, chewing, drooling, foaming, retching (abdominal contractions accompanied by oral gaping), and emesis (expulsion of stomach contents through the mouth) were recorded as quantal measures (presence or absence) during each 1-minute bin. The duration of hypersalivation was calculated by summing the total number of minute bins in which at least one of the following responses occurred: licking, chewing, drooling, and foaming. Dose-response functions were constructed by taking the mean (±SEM) duration of hypersalivation for each subject at each dose. The number of subjects to exhibit an emetic episode is reported for each dose. Total emetic episodes and latency to each emetic episode are also reported for doses that produced emesis in at least one subject. The same observer (LMW) documented all drug-induced responses in the present studies and was not blinded to treatment condition in order to respond efficiently to any untoward drug effects.

3 |. RESULTS

3.1 |. Assessment of drug-induced emesis and hypersalivation in squirrel monkeys

Figure 1 presents the emetic and hypersalivation effects following administration of 0.032–0.56 mg/kg nicotine (Figure 1A), 150–250 mg/kg LiCl (Figure 1B), 0.01–0.32 mg/kg arecoline (Figure 1C), and 0.032–0.32 mg/kg apomorphine (Figure 1D). Administration of 0.032 mg/kg nicotine failed to produce emesis in any of the four subjects. Administration of 0.1 mg/kg nicotine produced emesis in one subject, while larger doses (0.32 and 0.56 mg/kg) produced emesis in all four subjects. Administration of 150 mg/kg LiCl failed to produce emesis in any of the four subjects. Administration of 200 and 250 mg/kg LiCl produced emesis in three of four subjects. The fourth subject did not vomit during the 1-hour observation period following administration of 200 or 250 mg/kg; however, vomitus was noted in this subject’s home cage the morning after administration of both 200 and 250 mg/kg LiCl. Drug solubility and concerns of toxicity precluded the assessment of larger doses. Due to the challenge of quantifying responses outside of a 1-hour observation period, this subject was excluded from subsequent assessments of LiCl. Neither arecoline nor apomorphine, up to doses of 0.32 mg/kg, produced an emetic response in any subject. Assessment of a higher dose of arecoline (0.56 mg/kg) and apomorphine (1.0 mg/kg), which was conducted in a single subject, revealed no emetic episodes. Untoward observational drug effects following administration of these doses, including ataxia, prevented further examination of these or larger doses.

FIGURE 1.

Upper panels: Number of subjects to exhibit an emetic episode following administration of saline (S); 0.032–0.56 mg/kg nicotine, n = 4 (A); 150–250 mg/kg LiCl, n = 4 (B); 0.01–0.32 mg/kg arecoline, n = 4 (C); and 0.032–0.32 mg/kg apomorphine, n = 4 (D). Lower panels: Mean (±SEM) duration of hypersalivation (min) following saline or drug administration. Note that all ordinates are limited to 30 min to assist in magnitude comparisons across drugs

As with the emetic response, hypersalivation differed across drugs and dose. Both nicotine (Figure 1A) and arecoline (Figure 1C) produced dose-dependent increases in frequency and duration of hypersalivation. Administration of LiCl produced hypersalivation at all doses tested but not in a dose-dependent manner (Figure 1B). Apomorphine produced comparatively little hypersalivation across subjects (Figure 1D).

Following confirmation of the ability of nicotine and LiCl to produce emesis in the squirrel monkey, the episodic and temporal properties in emetic action of each drug were analyzed. Figure 2 summarizes the number of emetic episodes per subject (upper panels) and the latency to each emetic episode (lower panels) following emetic doses of nicotine (0.1, 0.32, 0.56 mg/kg) and LiCl (200, 250 mg/kg). In every instance across subjects, nicotine-induced emesis was limited to exactly one discrete episode (Figure 2A). The latency to the emetic episode following nicotine ranged from 2 to 11 minutes across subjects and dose (Figure 2C). The latency varied by subject, but not by dose—that is, each subject exhibited a characteristic latency (±2 minutes) across emetic doses. Unlike nicotine, LiCl intermittently produced multiple discrete emetic episodes (Figure 2B). In addition, larger variability in the latency to emetic episode was evident following LiCl administration relative to nicotine, with episodes ranging from 22 to 59 minutes following administration (Figure 2D).

FIGURE 2.

Upper panels: Total emetic episodes following administration of the emetic doses of nicotine (A) or LiCl (B). Bottom Panels: Latency to each emetic episode following administration of emetic doses of nicotine (C) and LiCl (D). Each symbol shape represents values from an individual subject (nicotine, n = 4; LiCl, n = 3)

3.2 |. Effects of pre-feeding on nicotine- and LiCl-induced emesis and hypersalivation

Figure 3 presents the effects of nicotine and LiCl on emesis (Figure 3A) and hypersalivation (Figure 3B) when administered 21 hours after feeding (black bars/circles) or 1hr after feeding (white bars/squares). Overall, 1-hour pre-feeding reduced the probability of emetic episodes following nicotine. Administration of 0.1 mg/kg nicotine 1 hour after feeding did not produce emesis in the subject that previously exhibited the emetic response at this dose 21 hours after feeding. In addition, administration of 0.32 mg/kg nicotine produced emesis in only one subject (instead of in all four) under pre-feeding conditions but did not alter the ability of 0.56 mg/kg nicotine to produce emesis in all subjects. The probability of LiCl-induced emesis was also reduced following pre-feeding at the lower emetic dose (200 mg/kg). However, pre-feeding increased the total number of emetic episodes following administration of 250 mg/kg LiCl (Figure 3B). Pre-feeding did not markedly alter hypersalivation produced by nicotine (Figure 3C) or LiCl (Figure 3D).

FIGURE 3.

Effects of pre-feeding on the total number of emetic episodes (A,B) and the duration of hypersalivation (C,D) following administration of nicotine (left panels) or LiCl (right panels). Subjects were fed either 21 h before (black bars, circles) or 1 h before nicotine administration (white bars, squares). Symbols to the left of the x-axis break of C and D represent the duration of hypersalivation following administration of saline (S). Each point in C and D represents the group mean ± SEM (nicotine, n = 4; LiCl, n = 3)

4 |. DISCUSSION

The present studies were conducted to determine the sensitivity of the squirrel monkey to four pharmacologically diverse emetic drugs. Nicotine and LiCl both reliably produced emesis, but with different profiles. Emetic doses of nicotine rapidly produced a single emetic episode, while administration of LiCl produced multiple emetic episodes with a timeframe that was considerably longer than that of nicotine. Only nicotine produced both emesis and dose-dependent hypersalivation, a prodromal sign often associated with emesis. Arecoline failed to produce emesis, but did produce dose-dependent hypersalivation. Apomorphine produced neither emesis nor hypersalivation.

One might assume that recent food intake would reliably modify the probability of vomiting following exposure to an emetic; however, a more complex and subtle interaction was observed. Pre-feeding attenuated the emetic effects of both nicotine and LiCl at lower doses, but did not diminish the emetic effect of the highest dose nicotine (0.56 mg/kg) and potentiated the effect of the highest dose of LiCl tested (250 mg/kg). Pre-feeding did not markedly alter nicotine- and LiCl-induced hypersalivation. Taken together, pre-feeding neither reliably served as a protectant to drug-induced emesis nor did it reliably exacerbate it.

Nicotine and LiCl are both commonly employed to elucidate mechanisms underlying emesis and to study the antiemetic effects of other drugs in laboratory animals.^{30,34,35,54,56,59} Nicotine has well-known emetic effects in humans.^26,60 Although we are not aware of any studies that have examined LiCl-induced emesis in humans, vomiting is an early sign of lithium toxicity in patients undergoing treatment for bipolar and other mood disorders with lithium carbonate or lithium citrate.⁶¹ Systemically administered nicotine most likely produces emesis primarily by acting at nicotinic acetylcholine receptors in the brainstem.^59,62 While the mechanisms of LiCl-induced emesis are less well characterized, it may act both in the brainstem and at the vagus and splanchnic nerves in the periphery.^63–65 Although documenting emesis beyond the 1-hour observation period following drug administration was outside of the scope of the present studies, vomitus was often found in a subject’s home cage during the 24-hour period following LiCl administration, but never following nicotine administration. These findings suggest (a) nicotine’s emetic profile in the squirrel monkey allows for precise elicitation of emesis, which could accommodate the appraisal of acute antiemetic drug efficacy of candidate therapeutics, and (b) LiCl is better suited for producing an emetic condition with an extended time course, which is consistent with its long-standing use in conditioned flavor avoidance and anticipatory nausea in rodents,^30,66 shrews,⁶⁷ and non-human primates.^64,68 In addition, differences in the emetic profiles of nicotine and LiCl suggest that hypersalivation may be prodromal to vomiting induced by some emetics (eg, nicotine) but not others (eg, LiCl); however, the underlying mechanisms responsible for the observed differences are currently unclear.

Arecoline failed to produce emesis but, nevertheless, serves as an interesting negative control. Emesis has been reported occasionally in humans⁶⁹ and in rhesus macaques⁷⁰ following administration of this muscarinic acetylcholine receptor agonist. Scopolamine, the prototypical motion sickness medication, is a muscarinic acetylcholine receptor antagonist, which would suggest that activity at muscarinic receptors could modulate an emetic response. However, the vestibular system is implicated in motion-induced emesis, but not necessarily in drug-induced emesis.⁷¹ Indeed, shrews selectively bred for motion sickness do not display a higher sensitivity to nicotine-induced emesis than wild-type shrews.⁷² Arecoline did produce hypersalivation in a dose-dependent manner, despite failing to produce emesis. High doses of arecoline also produce salivation in several other laboratory species, including rodents⁷³ and rhesus macaques.⁷⁰ In addition, medications with muscarinic agonist activity are often prescribed to treat xerostomia (dry mouth) resulting from radiotherapy and Sjogren’s syndrome.^74,75 Currently, it is unclear whether the salivation produced by arecoline in the absence of emesis is related to the prodromal hypersalivation that reliably precedes nicotine-induced emesis or if it represents a different response.

The effects of apomorphine in the present studies reiterate an important cautionary tale. As discussed above, apomorphine produces a reliable and rapid emetic response in humans,⁵¹ dogs,⁵⁰ cats,⁷⁶ ferrets,²⁶ and, interestingly, marmosets.⁵³ The squirrel monkey’s lack of emetic response to apomorphine, however, is consistent with other non-human primate laboratory species, including the rhesus and cynomolgus macaque.⁵²

The present studies provide the first systematic examination of drug-induced emesis in the squirrel monkey. The reliable and reproducible emetic response observed following administration of nicotine and LiCl indicates that the squirrel monkey is a suitable laboratory species in which to study this biological process. The evolutionary proximity of non-human primates to humans allows for the characterization of a comprehensive drug profile with potentially high translational value.³⁷ Moreover, the ability to incorporate emetic and antiemetic findings within behavioral, cognitive, and neural methodologies that are already well-established in the non-human primate could facilitate the development of safer and more effective pharmacotherapies. These advantages are particularly important in light of the fact that nearly all currently prescribed antiemetics (eg, ondansetron, nabilone, scopolamine) and also those under investigation (eg, cannabidiol) interact with complex neurotransmitter systems in the brain and have behavioral side effects, ranging from sedation or restlessness to cognitive disruption and, in rare cases, psychosis.^77,78 In addition, some currently available antiemetics appear to have abuse liability, for example, the antihistamine cyclizine, likely due to its stimulant effects.⁷⁹ Moreover, the antihistamine promethazine and NK-1 receptor antagonist aprepitant have both been reported to potentiate the euphoric effects of opioids,^80,81 increasing the likelihood of misuse. Therefore, the availability of an experimental subject in which the effects of a candidate antiemetic could be examined concurrently with assessments of cognitive disruption and abuse liability could accelerate the discovery of new pharmacotherapies that are both effective and have a reduced side-effect profile.

To further advance model development in non-human primates with an aim toward improved treatment options, validation of other emetic triggers will be required. For example, future research using squirrel monkeys could examine their responses to highly emetogenic chemotherapeutic agents in humans (eg, cisplatin, cyclophosphamide, dacarbazine) to determine whether a non-human primate model could effectively contribute to the development of improved prophylactics for chemotherapy-induced emesis. In the meantime, the present findings indicate that the squirrel monkey is sensitive to a variety of emetic triggers and, therefore, is well-positioned to serve as a laboratory subject in studies investigating emetic mechanisms and antiemetic drug development.